Dataset Information

CRISPR-mediated isolation of specific megabase segments of genomic DNA.

ABSTRACT: Megabase-sized, complex, repetitive regions of genomes are poorly studied, due to the technical and computational challenges inherent to both assembling precise reference sequences and accurately assessing structural variation across contiguous megabase DNA regions. Here we describe a strategy to overcome these challenges, CISMR (CRISPR-mediated isolation of specific megabase-sized regions of the genome), which enables us to perform targeted isolation of contiguous megabase-sized segments of the genome. Direct sequencing of the purified DNA segments can have >100-fold enrichment of the target region, thus enabling the exploration of both DNA sequence and structural diversity of complex genomic regions in any species.

SUBMITTER: Bennett-Baker PE

PROVIDER: S-EPMC5737698 | biostudies-literature | 2017 Nov

REPOSITORIES: biostudies-literature

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CRISPR-mediated isolation of specific megabase segments of genomic DNA.

Bennett-Baker Pamela E PE Mueller Jacob L JL

Nucleic acids research 20171101 19

Megabase-sized, complex, repetitive regions of genomes are poorly studied, due to the technical and computational challenges inherent to both assembling precise reference sequences and accurately assessing structural variation across contiguous megabase DNA regions. Here we describe a strategy to overcome these challenges, CISMR (CRISPR-mediated isolation of specific megabase-sized regions of the genome), which enables us to perform targeted isolation of contiguous megabase-sized segments of the ...[more]

PMID: 28977642

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Project description:BackgroundPichia pastoris (syn. Komagataella phaffii) is a widely used generally recognized as safe host for heterologous expression of proteins in both industry and academia. Recently, it has been shown to be a potentially good chassis host for the production of high-value pharmaceuticals and chemicals. Nevertheless, limited availability of selective markers and low efficiency of homologous recombination make this process difficult and time-consuming, particularly in the case of multistep biosynthetic pathways. Therefore, it is crucial to develop an efficient and marker-free multiloci gene knock-in method in P. pastoris.ResultsA non-homologous-end-joining defective strain (Δku70) was first constructed using the CRISPR-Cas9 based gene deficiency approach. It was then used as a parent strain for multiloci gene integration. Ten guide RNA (gRNA) targets were designed within 100 bp upstream of the promoters or downstream of terminator, and then tested using an eGFP reporter and confirmed as suitable single-locus integration sites. Three high-efficiency gRNA targets (PAOX1UP-g2, PTEF1UP-g1, and PFLD1UP-g1) were selected for double- and triple-locus co-integration. The integration efficiency ranged from 57.7 to 70% and 12.5 to 32.1% for double-locus and triple-locus integration, respectively. In addition, biosynthetic pathways of 6-methylsalicylic acid and 3-methylcatechol were successfully assembled using the developed method by one-step integration of functional genes. The desired products were obtained, which further established the effectiveness and applicability of the developed CRISPR-Cas9-mediated gene co-integration method in P. pastoris.ConclusionsA CRISPR-Cas9-mediated multiloci gene integration method was developed with efficient gRNA targets in P. pastoris. Using this method, multiple gene cassettes can be simultaneously integrated into the genome without employing selective markers. The multiloci integration strategy is beneficial for pathway assembly of complicated pharmaceuticals and chemicals expressed in P. pastoris.

Dataset Information

CRISPR-mediated isolation of specific megabase segments of genomic DNA.

Publications

CRISPR-mediated isolation of specific megabase segments of genomic DNA.

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